Interindividual variability in the beneficial and adverse effects of medications compromises safe and effective therapy. The long-term goal of this research is to understand the mechanisms that contribute to this variability. In the current funding period, we have focused on variability in drug disposition and response caused by CYP2B6. This hepatic enzyme metabolizes many clinically important drugs, but the clinical uses of these substrates are compromised by the extensive interindividual variability in CYP2B6 activity and its associated drug interactions. We discovered and validated clinically relevant novel substrate, inhibitors and genetic markers that now make CYP2B6 clinical research possible. Using these tools, we have demonstrated that CYP2B6 genetic variation not only affects substrate metabolism, but profoundly influences the degree of inhibition drug interactions in vitro and in vivo. This interplay between genotype and drug interactions is clinically important because an individual's genotype appears to affect dose adjustment needed for narrow therapeutic range CYP2B6 substrates (e.g., efavirenz and methadone) to avoid adverse effects. Despite these notable advances, the large portion of interindividual variability in CYP2B6 activity and associated drug interactions remains unexplained. In this competing renewal, we will capitalize on these novel and transformative clinical tools as well as exciting new data to comprehensively elucidate the mechanisms responsible for this variability in vivo. In Aim 1, we will test the hypothesis that the effect of nonsynonomous SNPs in CYP2B6 are substrate dependent and this, in turn, affects drug interactions. We will determine the influence of the CYP2B6*6 allele on kinetics of metabolism and inhibition of a panel of substrates and inhibitors in vitro. In Aim 2, we will determine how CYP2B6 genetic variants affect simultaneous auto- inhibition/autoinduction and collectively influence CYP2B6 activity. In vivo enzyme activity will be determined using bupropion as a probe at baseline (control), with a single 600 mg oral dose (inhibition) and after multiple doses (600 mg/day) (induction and inhibition) of efavirenz in healthy volunteers genotyped for CYP2B6*6 allele. A semi-PBPK model will be developed to predict the contribution each and interplay among these factors. In Aim 3, we will the test the hypothesis that genetic variants in genes that regulate CYP2B6 expression and function predict basal and drug-induced CYP2B6 activity. This association will be tested clinically using DNA, plasma and urine samples from our proposed (Aim 2) and previously completed efavirenz clinical trials. Our pathway-guided approach is expected to identify novel genetic biomarkers of interindividual variability in CYP2B6-mediated drug clearance and interactions, and may serve as a paradigm for studying other genes involved in drug metabolism. Overall, we will develop a comprehensive understanding of mechanisms responsible for variable CYP2B6 activity and the metabolism of its substrates, and this information could be eventually used to personalize therapy.